Transcript Document 7160913

分子分型在医院感染控制
中的作用
（the Role of Molecular
Typing in Nosocomial
Infection Control）
瑞金医院临床微生物科
瑞金医院医院感染办公室
杨 莉
分子分型（分子流行病学研究）
 从核酸分子水平上分析医院感染的发生、发展规律及机理，
更加准确有效地进行医院感染管理控制，已成为当前国际
医院感染管理研究中的重要方向。
 从患者分离株到病区周围环境株的比较分析；
 从外源性感染到内源性感染
 从某一医院的医院感染暴发到大范围甚至世界
范围的感染菌株流行变迁；
基因多态性分析技术已成为医院感染监测控
制的高水平研究领域。
2
微生物室在医院感染中的作用
 accurately identifying
nosocomial
pathogens
 detecting unexpected
antimicrobial-drug
resistance
 epidemiologic typing
 鉴定
 特殊耐药菌的
检出
 流行病学分型
Pfaller MA et al. The clinical microbiology laboratory and infection
control: emerging pathogens, antimicrobial resistance, and new
technology. Clin Infect Dis 1997;25:858-70.
3
分子分型
基因分型
分子流行病学
研究
 PFGE(脉冲场凝胶电
泳)
 RAPD(随机扩增DNA
多态性)
 REA(限制性酶切)
 ribotyping (核糖体分
型)
4
1、脉冲场凝胶电泳(PFGE)
制
备
琼
脂
糖
栓
块
消
酶
化
切
脉
冲
电
泳
结
果
解
释
5
PFGE原理
消化、释放出DNA
6
PFGE原理
Enzyme
酶切
约5～20个、长度
10～800kb的大片段
DNA
7
8

Tenover FC.et al.J Clin Microbiol,1995;33(9):2233～2239
PFGE原理
脉冲电泳
9
原理：
常规凝胶电泳
DNA
脉冲场凝胶电泳
10
PFGE原理
染色、拍照
时间消耗
 从分离菌株到出结果平均
2.5 天
 标本准备、细胞裂解－－第
一天
 酶切－－第二天
 染色、拍照－－第三天
11
12
PFGE结果判读
 目测法：
 按美国疾病控制和预防中心(CDC)Tenover等人推荐的方
法判读。
 图谱完全相同的定为一个型，彼此之间相差一个带的定
为同一型的不同亚型，相差2－3个带的认为亲缘关系密
切，相差4－6个带的认为可能相关，条带相差7个以上的
认为无亲缘关系。并随机地选择不同的字母如A、B、C、
D等的字母顺序分型。
 聚类分析
 计算机输入SPSS，做树状图
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PFGE同源性分析
D ic e ( O p t:1 .5 0 % ) ( T o l 1 .5 % - 1 .5 % ) ( H > 0 .0 % S > 0 .0 % ) [0 .0 % - 1 0 0 .0 % ]
O X AP C R
C T XP C R
P C R T e s tin g
SH V PC R
PFGE - XbaI
TE MP C R
100
80
60
PF GE - XbaI
. E X .0 0 4 2
E
01 A
. E X .0 0 4 4
E
07 A
. E X .0 0 4 5
E
07 A
. E X .0 0 3 8
E
01 A
. E X .0 0 3 8
E
01 A
. E X .0 0 3 8
E
01 A
. E X .0 0 1 3
E
01 A
. E X .0 0 4 0
E
01 A
. E X .0 0 1 5
E
01 A
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PFGE特点：
 DNA原位提取法，减少了断裂
 利用细菌全基因组信息
 细菌分型金标准
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2、RAPD（随机扩增DNA多态性）
 1990年
 Williams：
RAPD
 Welsh：AP-PCR
 本质上相同
 引物：
 “短” 、“单一” 和
“非特异性”，一般9～
10bp
 扩增条件：“非严格性”
 退火温度一般较低,25～
35℃
16
 ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129～1135
17

ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129～1135
18

ESHWAR MAHENTHIRALINGAM.et al.J Clin
Microbiol,1996;34(5):1129～1135
19
分子分型在医院感染控制中
的作用
20
Special Issue
New Technology for Detecting MultidrugResistant Pathogens in the Clinical
Microbiology Laboratory
Lance R. Peterson*† and Gary A. Noskin*†
*Northwestern Memorial Hospital and
†Northwestern University Medical School,
Chicago, Illinois, USA
EID, 2001,
7: 30621
 Northwestern Memorial Hospital,




Chicago
700-bed, university-affiliated medical
center
出院：>39,000/年
急诊：56,000例/年
门诊量：260,000/年
22
分型确认后及干预效果
5.79
P=0.002
LR Peterson
et al, EID,
2001, 23
7: 306
指标
1. the total number of nosocomial infections
per 1,000 patient days每千住院日医院感染
数
2. the number of patients with nosocomial
infections per 100 patient discharges
每100出院病人医院感染病人数
(percentage of patients with nosocomial
infection)
（医院感染病人百分比）
24
感染控制工作小组成员：
 We formed a permanent, integrated infection
control and prevention program that fully
incorporates




infection control personnel,
infectious disease personnel,
pharmacy personnel,
clinical microbiology
personnel
into a single working group to
minimize hospital infections.
 感染控制
 感染性疾病
 药学
 临床微生物
Hacek DM et al. Am J Clin Pathol 1999;111:647-54.
25
资料收集方法（Methods for data
collection）：
 review of microbiology reports
 review of patients' medical
records,
 direct observation of medical
and nursing practice,
 active surveillance of rectal
cultures of patients in nursing
units for high-risk patients,
 evaluation of suspected
nosocomial infections reported
by health-care providers.
 查阅微生物报告
 查阅病史记录
 对医护人员操作的直接
观察
 高危病人直肠培养的动
态连续观察
 对上报的可疑医院感染
的评估鉴定
由三位专职感染控制人员对资料进行汇总分析，并制
定出相应控制措施，并在医院感染控制和预防部门主管的指
导下实施。
26
Two interventions：
 a molecular typing
laboratory
 a weekly planning
meeting
 infection control
 diagnostic medical
microbiology (molecular
epidemiology)
 Pharmacy
 and infectious diseases
两个主要的干预措施：
 分子分型
 周会
包括以下方面的代表：
 感染控制
 微生物诊断（分子
流行病学）
 药学
 感染性疾病
27
weekly meetings：
周会内容：
 the ongoing short- and long 医院感染动向（短
term trends in nosocomial
期、长期）
infections within the center
 activities of the infection control  感染控制专职人员
professionals and microbiology
和微生物实验室的
laboratory personnel;
工作
 any needed changes were
determined.
 决定需要做的调整
 The organizational structure for  需分型的病原体与
selecting microbes for typing
主管讨论决定
was shared by the medical
directors of infection control and
clinical microbiology
28
需基因分型的微生物：
 routinely genomically typed：VRE
 Periodic routine typing：
 fluoroquinolone-resistant P.
aeruginosa,
 methicillin-resistant Staphylococcus
aureus (MRSA),
 Enterobacter cloacae,
 Clostridium difficile
 Additional organisms for typing：
selected through surveillance of
microbiology culture reports
discussed at the weekly meeting.
 常规基因分型：VRE
 周期性分型：
 氟喹诺酮耐药的铜绿
 MRSA
 阴沟肠杆菌
 难辨梭菌
 其他：根据微生物报告
并在周会上讨论后决定
需基因分型的病原体
任何时候工作小组要求进行基因分型，临床微生物
29
实验室即将菌株提供给分子分型部门并执行
实验方法
REA analysis
restriction of genomic DNA with
conventional electrophoresis
DNA限制性酶切
30
成本-效益分析（Analysis of Cost Data）
基因分型相关费用
 the cost of equipment,
remodeling,
 reagent and other
supplies,
 salaries and benefits
for three technologists,
 plus all the institutional
assessments (e.g., fullcost basis) required to
operate a hospital
laboratory.
 仪器配制及维修、实
验室改造等；
 试剂及其他消耗品
 三位实验员的薪水
 所有的评估分析（如
full-cost basis ）需
要动用全院系统
分析方法：t检验
31
结果：
 1、VRE
 initial impetus: serious

nosocomial problem--VRE's emergence
molecular typing results:
 a pattern of numerous
“mini” patient-to-patient
outbreaks of distinct
clones
 rather than the spread of
a single persisting strain
 1、VRE
 最初调查：VRE医
院感染严重
 分型结果提示：
 多型别、小规模
（mini）病人间流
行
 而不是一个型别的
流行
32
结果：
 genomic typing:
 基因分型：可将可能的医院
 patient-to-patient
transmission;
 nosocomial outbreak;
 little evidence of
horizontal spread
 Using this information, we
determined what
intervention was likely to
control an apparent
outbreak (20).

感染分组：
 病人之间交叉感染(high
conality, >90%)
 感染爆发(moderate
clonality, 35%-75%)
 无水平传播(<20%
clonality).
在此基础上，决定采取哪种
控制措施
33
high
conality,
>90%
moderate
clonality,
35%-75%
clonality,
<20%
likely
possibly
unlikely
patient-topatient
transmission
nosocomial
outbreak
little evidence
of horizontal
spread
similarity
34
 During the last 2
years of this study,
 25 possible microbial
outbreaks were
investigated by the
typing laboratory
 VRE,
 fluoroquinoloneresistant P. aeruginosa,
 MRSA,
 E. cloacae,
 C. difficile.
 通过基因分型，共鉴别
25起微生物感染爆发
 VRE
 氟喹诺酮耐药的铜绿
 MRSA
 阴沟
 难辩梭菌
35
 Classic Spread of Nosocomial 典型的医院感染传播
Infection
 VRE: 19 strains, from 16 patients, in
a 2-month period;
 14 strains: from one of two clones
(88%)
 Indicating: a high probability of
nosocomial spread
 Review:
 检查分析:
 microbiology laboratory: culture
requisitions---no close contact.
 Patients: existing direct connection
between 11/14 patients (14).
 infection control practices:
aborted the outbreak
 VRE: 19株, 来自16个病
人，2个月时间内;
 其中十四株：为两个型
别中的一个型别 (88%)
 高度提示感染传播
 微生物实验室: 培养过
程无密切联系
 患者：14人中有11人有
直接联系
 感染控制：中止暴发
36
Moderate Likelihood of Spread of
Nosocomial Infections
 During a 1-month period, in a special- 较有可能为NI传播
 1个月时间内，特殊病房
care unit
 invasive infections, caused by five  侵入性操作感染：
 肺炎克雷伯菌
isolates
 表皮葡萄球菌
 Klebsiella pneumoniae,
 溶血葡萄球菌
 S. epidermidis, and
 40%-60% clonality
 S. hemolyticus
 DNA typing indicated 40% to 60% for  分析：分离出相同型别菌株的
each of the bacterial species.
 patients with genetically identical
organisms occupied adjacent beds.
 Erecting a barrier on the unit, along
with educating medical staff, halted
the spread of these infections (15).
患者病床临近
 措施：
 病房设立屏障
 医护人员教育
 结果：感染中止
37
Outbreaks not Caused by Patient-toPatient Spread
 Suspected outbreaks
consisting of four isolates of K.
pneumonia and 64 strains of
Serratia marcescens were
investigated in the ICUs of two
hospitals. Both investigations
showed 21% clonality,
indicating unlikely patient-topatient spread.
 Investigation suggested
suboptimal handling of
ventilator equipment, and both
outbreaks were stopped by
retraining of personnel using
this equipment
 2个医院的ICU病房
 4株肺克，64株粘质沙雷





菌
21% clonality
提示：非病人之间传播
indicating unlikely
patient-to-patient
spread.
调查分析：机械通气相关
操作不规范
措施：规范操作
结果：感染中止
38
 Pseudooutbreaks
 Possible outbreaks occurred in the




 2个医院，特殊护理病
special-care nursery units of two
hospitals, each of which had its own
molecular typing section.

seven S. aureus strains, and the other
of four isolates of gram-negative bacilli. 
immediately typed and no (20%)
clonality existed.

No interventions were instituted, and 
the apparent outbreaks were

determined to be normal variation in
infections (15,21).
avoided culture-based surveillance
investigation of staff by the state
department of health, and the other
avoided closing the unit for a 30-day
full disinfection and cleaning (done in
previous suspected outbreaks).
房，每个医院都有自
己的分子分型部门
7株金葡 4株G-菌
迅速分子分型rapid
typing
no (20%) clonality
没有采取措施
节省：
 医护人员携菌情况
调查（培养）
 关闭病房30天，消
毒、清洁
39
分型确认后及干预效果
4
Typing Intervention
3.5
3.3
3
2.56
2.5
NI/100 Patients
2
1.5
1
0.5
P=0.000006
0
1993
1994
1995
1996
1997
1998
1999
LR Peterson
et al, EID,
2001, 40
7: 306
分型确认后及干预效果
5.79
P=0.002
LR Peterson
et al, EID,
2001, 41
7: 306
 nosocomial infection:



3.3%-2.6% (national rate:
4.4%-5%)
>1,400 fewer patients
acquired infections
during this time,
averting more than 50
expected deaths
Even with endemic VRE,
most of our outbreaks
involve three or fewer
patients (19).
 医院感染：3.3%下降
至2.6%(全国医院感染
率：4.4%-5%)
 减少>1,400的病人感
染
 死亡：减少了>50
 VRE：涉及的病人也比
其他医院少
42
成本
The mean number of
医院感染患者数量平
patients with nosocomial
infections decreased by 283 均每年下降 283，住
per year, a reduction of
院天数下降超过1100
more than 1,100 inpatient
天
days.
The costs avoided by
因此节省的费用平均
using this calculation
averaged more than
$2,150,000/year, based on
1999 dollars.
每年超过$2,150,000
（与1999年相比）
43
 Representatives now meet



together for 45 minutes
each week
For Microbiology, opening
the typing laboratory totaled
$180,050. By the fifth year,
costs in the laboratory
section were stable.
The cost for the laboratory,
includng three medical
technologists, is $400,000
yearly.
Virtually all these costs are
borne by the hospital.
 小组聚会逐渐转为每
周开会，45min，讨
论
 微生物室成立分子分
型实验室（设备及人
员）的费用为
$180,050.
 每年分子分型相关支
出为$400,000
 医院承担
44
 While such a grant


program would cost up
to $2 billion each year
if all U.S. hospitals
participated,
the projected reduction
in cost of treating
nosocomial infections
could reach over five
times that amount.
a savings of $5.00 for
each dollar spent.
 假设：美国所有医
院
 进行基因分型相关
费用达到20亿美元
 节省下来的治疗医
院感染的费用将超
过5倍（100亿）！
 每使用1美元节省5
美元
45
 Typing time: within 1 week

48 hours.
Lack of clonality: suggests
other reasons for the
apparent outbreak,
 antimicrobial-agent use
pressure,
 failure of appropriate nursingcare practices,
 or simply random variation in
the number of infections.
 时间：1周，48h
 如没有流行相关线索，
可能是其他原因：
 抗生素压力,
 护理操作不当（非感
染相关）,
 仅仅是感染数量的随
机变化
 Early knowledge of whether  早发现、及早确定调
microbial clonality is present
查范围、采取合适的
or absent focuses the scope
干预措施
of an investigation and
facilitates appropriate
intervention.
46
 cost of rapid detection using
the polymerase chain reaction
(PCR)
 =one day of glove isolation
 could be completed in a single 8hour workday.
 As gene chip technology
moves into clinical use,
detecting a large number of
resistance determinants soon
after a patient is admitted to
the hospital should be
possible.
 PCR 分型
 费用=一天的手套
费用
 8小时的工作时间
内可完成
 基因芯片：
 大规模
 耐药监测
 病人入院后即实施
47
分子分型在医院感染中的应用：
 technically possible
 medically useful
 economically justified
48
 医院感染的分子流行病
学研究方法
 分子流行病学方法在医
院感染中的应用
 医院感染控制的人员安
 临床医院感
染控制
 科研
排
 成本-效益研究
49
实验研究：
 瑞金医院 04年～05年
 全耐药鲍曼不动杆菌（PRAB） 各个科室的

突然增多
经脉冲场凝胶电泳（PFGE）证实
 烧伤科为单独一个型别PRAB科室内流行
 除烧伤科以外的其他科室为科室间同一型别
PRAB流行。
 由此可见，分子流行病学方法：
 为医院感染控制提供准确的实验数据
 有效判断感染来源和流行趋势
 为更好的做好医院感染控制工作打下了基础。
50